Processes for identifying a solvent condition suitable for determining a biophysical property of a protein

ABSTRACT

The present invention provides processes for identifying a buffer condition suitable for determining a biophysical property of a protein. The processes of this invention utilize vapor diffusion means to alter buffer conditions, thus minimizing the volume of test samples and thereby conserving protein material. The processes of this invention are particularly useful to determine a buffer condition suitable for performing NMR studies on a protein.

TECHNICAL FIELD OF THE INVENTION

The present invention provides processes for identifying a buffercondition suitable for determining a biophysical property of a protein.The processes of this invention utilize vapor diffusion means to alterbuffer conditions, thus minimizing the volume of test samples andthereby conserving protein material. The processes of this invention areparticularly useful to determine a buffer condition suitable forperforming NMR studies on a protein.

BACKGROUND OF THE INVENTION

Elucidation of the biophysical properties of a protein in solution is akey to understanding its biological activity. Numerous techniques havebeen developed in the prior art to characterize a protein in solution.They include assays to determine functional activity, immunoreactivityand protein concentration, spectral methods such as Ultra-Violet,Visible, Infra-Red and fluorescence spectroscopy, Circular Dichroism,light scattering, surface plasma resonance, calorimetry, NuclearMagnetic Resonance, High-Pressure Liquid Chromatography, gelelectrophoresis, terminal sequencing analysis, and Mass Spectrometry.Such techniques typically monitor a biophysical property of the proteinin solution, such as solubility, activity, ligand binding, aggregationstate, and the conformation or folding state.

For example, spectral methods, such as Nuclear Magnetic Resonance (NMR),Circular Dichroism, fluorescence and absorbance spectroscopy providedetailed information regarding the secondary and tertiary structure ofproteins in solution, changes in the behavior of a protein underdifferent solvent conditions, comparison of properties between theprotein and homologous or mutated forms of the protein, stability of theprotein in solution, and structural transitions such as unfolding andrefolding under a variety of conditions.

One of the major limitations in studying a biophysical property of aprotein by the above techniques is the solubility and stability of theprotein in solution. For example, NMR studies of proteins typicallyrequire a concentrated protein solution that is stable enough foracquisition of data for several days of more. This requirement hasproven to be extremely difficult to satisfy on a consistent basis, andmany NMR studies have been delayed or abandoned because of inability tostabilize or solubilize the protein of interest. This factor is likelyto become more of a limitation in the future as NMR spectroscopistsattempt to study larger proteins.

Typically, a solvent condition for a protein comprises a buffer systemthat maintains the pH of the solution at or near a constant value and,optionally, contains stabilizers such as salt, detergent, glycerol andexcess reductant. The solvent condition in a protein solution is morecommonly described as the buffer condition because a solution containinga protein almost always contains a buffer system.

At present, the main approach for identifying buffer conditions forstudying the biophysical properties of a protein consists oftransferring the protein at relatively low concentration into solutionswith various buffer and pH conditions, and then concentrating thesolutions and assessing the solubility and stability. Once a buffer andpH have been identified in which the protein is soluble and reasonablystable, an empirical approach is taken to varying stabilizers such assalt, reducing agents, glycerol, detergents, etc., in order to maximizesolubility and stability.

In addition to enhancing solubility and stability, a stabilizer shouldnot interfere with the technique employed to study the biophysicalproperty. For example, if a protein solution is to be studied by NMRspectroscopy, then any stabilizer present in the solution should notgive rise to resonances that interfere with the NMR spectrum. As anotherexample, if a protein solution is to be studied by ultra-violetabsorbance spectroscopy, then any stabilizer present in the proteinsolution should not strongly absorb ultra-violet radiation in the samefrequency range as the protein.

The empirical approach for identifying appropriate buffer conditions isinefficient because the step of transferring the protein into variousbuffer systems is tedious and time-consuming. Moreover, sampling all ofthe possible combinations of buffer conditions consumes large amounts ofvaluable protein.

This is a crucial limitation for proteins that are extremely difficultto isolate and purify.

Thus, there is a need for a method to rapidly and efficiently identify abuffer condition in which a protein is soluble and stable. Such a buffercondition would be highly suitable for determining a biophysicalproperty of a protein in solution using any one of the known techniques.

X-ray crystallographers have long had to contend with the converseproblem in identifying conditions for precipitating a protein out ofsolution in order to grow crystals. Currently, the technique of vapordiffusion [1] is used to carry out controlled precipitation, and iscombined with incomplete factorial [2] or sparse matrix [3] methods inorder to screen large matrices of solvent conditions.

In a typical vapor diffusion method, a protein solution is combined witha precipitating agent and the mixture is sealed within a chambercontaining a solvent reservoir in such a way that the solvent isgradually drawn out of the protein solution, leading to supersaturationand precipitation of the protein crystal.

The present invention provides a process for identifying bufferconditions that are suitable for determining a biophysical property of aprotein in solution. The process of the present invention uses vapordiffusion, but differs from the crystal growing technique discussedabove in that the absence of precipitants and the optional presence ofstabilizers allows the optimization of solubility instead ofinsolubility.

SUMMARY OF THE INVENTION

The present invention provides a process for identifying a buffercondition suitable for carrying out a study for determining abiophysical property of a protein.

The process of this invention comprises the steps of:

a. providing a protein dissolved in a first buffer condition;

b. modifying said first buffer condition to a second buffer conditionthrough vapor diffusion means; and

c. evaluating the suitability of said second buffer condition fordetermining the biophysical property of the protein.

The process of this invention can be utilized to either concentrate ordilute a protein in solution, as well as to test the behavior of aprotein in different concentrations of buffering salts and otherpH-maintaining reagents, other salts and/or stabilizers. Different typesof conditions are desirable depending upon the biophysical property ofthe protein to be determined. The processes of the present inventionadvantageously allow numerous different buffer conditions to be testedwithout wasting large amounts of potentially precious protein during thetesting process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-section view of the apparatus used for thehanging-drop method of the present invention.

FIG. 1B is a cross-section view of the apparatus used for thesitting-drop method of the present invention.

FIG. 1C is a cross-section view of the apparatus used for thesandwich-drop method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following abbreviations are used:

NMR--Nuclear Magnetic Resonance

MES--[2-(N-morpholino)ethanesulfonic acid]

HEPES--N-2-hydroxyethylpiperazine-N-2

TRIS--[tris-(hydroxymethyl)-aminomethane]

DMSO--dimethyl sulfoxide

CHAPS--3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate

BOG--n-hexyl-b-D-glucopyranside

ACES--(N-2-acetamido-2-aminoethane-sulfonic acid)

BES--[N,N-bis-(2-hydroxyethyl)-2

Bicine--[N,N-bis-(2-hydroxyethyl)-glycine]

BIS-Tris--{[bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethyl)-methane}

BIS-Tris-propane--{1,3-bis-[tris-(hydroxylmethyl)-methylamino]-propane}

CAPS--[3-(cyclohexylamino)-propane-sulfonic acid]

CHES--[2-(N-cyclohexylamino) ethanesulfonic acid]

HEPES--(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)

MES--[2-(N-morpholino) ethanesulfonic acid]

MOPS--[3-(N-morpholino) propanesulfonic acid]

PIPES--[piperazine-N,N'-bis-)2-ethanesulfonic acid]

TAPS--(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid)

TES--(2-{[tris-(hydroxymethyl)-methyl]-amino}-ethanesulfonic acid)

Tricine--{N-{tris-(hydroxylmethyl-methyl]-glycine}

The present invention provides a process for identifying a buffercondition suitable for determining a biophysical property of a protein.According to one embodiment, the process comprises the steps of:

a. providing a protein dissolved in a first buffer condition;

b. modifying said first buffer condition to a second buffer conditionthrough vapor diffusion means; and

c. evaluating the suitability of said second buffer condition fordetermining the biophysical property of the protein.

The term "buffer condition", as used herein denotes a solution capableof maintaining the pH at or near a constant value. A buffer conditioncomprises a solvent, a reagent capable of maintaining pH, and theprotein dissolved therein. Additional components, such as a stabilizer,may also be present in a buffer condition if they are necessary.

Buffer conditions for proteins are well known in the art, and typicallycomprise water as a solvent and an acid-base conjugate pair to maintainpH. Organic solvents and other volatile solvent may also be utilized,but are less desirable.

Reagents capable of maintaining pH that are useful in the bufferconditions of the present invention include, but are not limited to,potassium phosphate, sodium phosphate, sodium acetate, sodium citrate,ammonium acetate, cacodylic acid, imidazole, boric acid, bicine, ACES,BES, BIS-Tris, BIS-Tris-propane, CAPS, CHES, glycine amide,glycylglycine, MES, MOPS, PIPES, HEPES, TAPS, TES, tricine,triethanolamine, or TRIS. Preferred pH-maintaining reagents arepotassium phosphate, sodium phosphate, sodium acetate, ammonium acetate,cacodylate, imidazole, bicine, BIS-Tris, sodium citrate, imidazole, MES,MOPS, PIPES, HEPES, triethanolamine, or TRIS.

According to one embodiment, the process of the present inventioncomprises the first step of providing a desired protein dissolved in afirst buffer condition. This step may be achieved in a number of ways.For example, a protein may be directly dissolved in the first buffercondition.

Alternatively, the protein may be dissolved initially in a solution thatcontains some, but not all, of the components of the first buffercondition. Once the protein is dissolved in such a solution, theremaining first buffer condition components may then be added to theprotein solution, either from concentrated solution stocks or as solids.Yet another method for providing a protein dissolved in a first buffercondition is to first dissolve the protein in a solution and thendialyze that protein solution against another solution, such that uponcompletion of dialysis, the protein is dissolved in the first buffercondition. Other alternatives to direct dissolution of the protein intothe first buffer condition include initial dissolution of the proteininto a solution followed by buffer exchange into the first buffercondition using gel filtration, centrifugal filtration, orultrafiltration methods, all of which are well known in the art.

Preferably, the first buffer condition comprises a minimum concentrationof the components (i.e., pH-maintaining reagents, stabilizers) that arecapable of maintaining the pH at or near a constant value and of keepingthe protein dissolved.

Stabilizers may also be utilized in the first buffer condition whendissolution of the protein is otherwise difficult. Stabilizer which maybe useful in the methods of this invention include salts, reducingagents, detergents, glycerol, polyols, osmolytes, chaotropes, organicsolvents, electrostatic reagents, metal ions, ligands, inhibitors,cofactors or substrates, chaperonins, redox buffers, disulfideisomerases and protease inhibitors.

Typically, it is desirable that the stabilizer concentration in thefirst buffer condition is kept low in order to avoid potential problems.For example, a large excess of reducing agents may reduce structurallyimportant disulfide bonds; glycerol viscosity effects may induceunacceptable line broadening in NMR spectroscopy; and detergents mayform micelles above a critical concentration.

The second step in the method of this invention is the modification ofthe first buffer condition into a second buffer condition which isachieved through vapor diffusion means. The second buffer conditiondiffers from the first buffer condition in any or all of the followingfeatures: changes in the concentration of the protein, changes in theconcentration of reagents that maintained pH in the first buffercondition, changes in the concentration of stabilizers that may havebeen present in the first buffer system, and changes in pH. The transferof solvent due to vapor diffusion modifies the first buffer condition toa second buffer condition.

According to one embodiment of the present invention, the vapordiffusion means for modifying the first buffer condition consists of aphysically closed system first comprising a chamber containing areservoir solution and a vessel that holds the protein dissolved in thefirst buffer condition.

The vessel that holds the protein dissolved in the first buffercondition may be a cuvette, a test tube, a multi-well plate, or anyother suitable vessel. Alternatively, the vessel may be a slide or coverslip which has a surface tension sufficient to keep a droplet of proteindissolved in the first buffer condition attached to its surface evenwhen the vessel is inverted ("hanging drop").

The reservoir solution comprises a solvent. That solvent may be eitheraqueous or organic in nature. The solvent may further comprise any orall of the following dissolved therein: acid or base, salts,pH-maintaining reagents, and stabilizers.

The reservoir solution must also have a vapor pressure sufficient toallow vapor diffusion of a solvent between the reservoir solution andthe first buffer condition. As will be apparent to those in the art, thevessel containing the first buffer condition must be physicallypositioned with respect to the reservoir solution so as to allow vapordiffusion to occur. This is preferably achieved by forming a closedsystem comprising the reservoir in close physical proximity to thevessel holding the first buffer condition.

Vapor diffusion techniques known in the prior art can be readilyemployed in the processes of the present invention. Such techniquesinclude, but are not limited to, the hanging drop method, the sittingdrop method and the sandwich-drop method.

For example, in the hanging-drop method, a protein solution in a firstbuffer condition, typically 0.5-8 mL by volume, is placed on a glassslip. The glass slip is then inverted and sealed onto a well containinga reservoir buffer solution having a vapor pressure different from thatof the protein solution. A schematic of a typical apparatus for thehanging-drop method is shown in FIG. IA. The exposure to the reservoirsolution results in vapor diffusion of solvent into or out of theprotein solution. This diffusion modifies the first buffer condition ofthe protein solution to a second buffer condition.

In the sitting-drop method, a drop of protein solution in a first buffercondition, typically up to approximately 200 mL by volume, is placed ona plate having a depression to hold the protein solution. The plate isthen placed in a chamber and exposed to a reservoir buffer solutionhaving a vapor pressure different from that of the protein solution. Aschematic of a typical apparatus for the sitting-drop method is shown inFIG. IB. The exposure to the reservoir buffer solution results in vapordiffusion of solvent into or out of the protein solution. This diffusionmodifies the first buffer condition of the protein solution to a secondbuffer condition.

In the sandwich-drop method, a drop of protein solution is sandwichedbetween two glass plates and sealed in a chamber containing a reservoirsolution having a vapor pressure different from that of the proteinsolution. A schematic of a typical apparatus for the sandwich-dropmethod is shown in FIG. IC. The exposure to the reservoir buffersolution results in vapor diffusion of solvent into or out of theprotein solution. The diffusion modifies the first buffer condition ofthe protein solution to a second buffer condition. Those of skill in theart will recognize that salts and pH-maintaining reagents will be themain contributors to the osmolarity of the various solutions and bufferconditions utilized in this invention. This is because these reagentswill normally be present in much higher concentration than the proteinor other stabilizers.

According to one embodiment, the solvent in the reservoir solution vapordiffuses into the protein solution with the first buffer condition,thereby increasing the amount of solvent in the protein solution; i.e.,diluting the components present in the first buffer condition. Such adilution occurs when the osmolarity of the first buffer system is lowerthan that of the reservoir solution.

The dilution method is particularly useful for identifying alternatestabilizers ("test" stabilizers) for proteins that have heretoforerequired the presence of a chaotrope or a high concentration ofstabilizer--both of which were undesirable for studying a biophysicalproperty of the protein. The desired test stabilizer is one thatstabilizes the protein without precipitating it, while being present inlow enough concentration to not interfere with subsequent studies of abiophysical property of the protein.

This may be carried out by creating first buffer conditions through theaddition of different "test" stabilizers and/or different amounts of"test" stabilizers to protein solutions containing the undesirablechaotrope or stabilizer. The resulting plurality of first bufferconditions are then exposed to a low osmolarity reservoir solution underconditions which allow solvent to diffuse into the protein solution.This creates a second buffer condition wherein the concentration of theundesirable chaotrope or stabilizer is reduced to below the levelnormally needed to keep the protein soluble. Each second buffercondition is then analyzed to determine if the "test" stabilizer iseffective in keeping the protein soluble. The solubility of the proteinin the second buffer condition can be readily determined by examiningeach drop for clarity and an absence of precipitate. If the proteinremains soluble in the second buffer condition, one may then performtests to determine whether additional biophysical properties of theprotein can be assayed in that second buffer condition.

Because the dilution method described above uses small volumes ofprotein solution (as little as 10 to 100 ul), the method can be readilyextended to a batch screening method using a 96-well microtiter plate.Each of the 96 wells in the plate is filled with a drop of proteinsolution having a different first buffer condition (varying types andconcentration of "test" stabilizer). The plate is then subjected tovapor diffusion in the presence of a reservoir solution. The advantageof using a microtiter plate is that it lends itself readily toautomation of the solubility assay.

In addition to screening the efficacy of new stabilizers, the effect ofchanging the pH of the protein solution may also be readily evaluated bythe dilution method. In such a case, the reservoir solution contains avolatile acid or base. As the acid or the base diffuses into the drop ofprotein solution, it creates a second buffer condition having adecreased or increased pH, respectively.

According to another embodiment, solvent diffuses out of the firstbuffer condition into the reservoir solution, thus creating a secondbuffer condition wherein the reagents contained in the first buffercondition have a higher concentration. Such a diffusion of solventoccurs when the osmolarity of the first buffer system is less than thatof the reservoir solution.

This concentration method may be employed to simultaneously test aplurality of second buffer conditions to determine in which one aprotein is most soluble. For example, in one particular embodimentindividual aliquots (preferably less than 100 μl and more preferablyless than 10 μl) of a protein solution are combined with differentpH-maintaining reagents and/or stabilizers to create a plurality offirst buffer conditions. Thus, the various first buffer conditions maydiffer from each other in the concentration of protein, the type orconcentration of pH-maintaining reagents, the pH, and/or the type orconcentration of stabilizer.

Each of the plurality of first buffer conditions is then exposed to areservoir solution of higher osmolarity under conditions which allowsolvent to diffuse out of the first buffer conditions into the reservoirsolution. This modifies the first buffer condition of each proteinsolution to a second buffer condition wherein the concentration ofprotein (and the other reagents) is increased.

For example, the vapor diffusion method described above can be carriedout by the hanging drop method using a Linbro plate with 24 wells. Thus,24 first buffer conditions, each differing in the buffer type orconcentration, protein concentration, pH, and/or stabilizer type orconcentration, are prepared and a drop of each is placed on a separateglass cover slip. In a preferred embodiment, the particularpH-maintaining reagents in the reservoir solution and the first buffercondition are the same, although the concentration of those reagents islower in the first buffer condition.

Each well is then partially filled with a reservoir solution having ahigher osmolarity than the first buffer condition. The glass cover slipcontaining the first buffer condition is then inverted and sealed ontothe well containing the corresponding reservoir buffer. The plate isthen left undisturbed to allow vapor diffusion to occur, and the bufferconditions of each of the 24 drops is modified to a second buffercondition.

If the pH-maintaining reagents in the reservoir solution and the firstbuffer condition are identical, but lower in concentration in thelatter, solvent will diffuse out of each drop until the totalconcentration of buffer in the drop is approximately the same as theconcentration in the reservoir (assuming that the pH-maintainingreagents are the main contributor to the osmolarity of both thereservoir solution and the first buffer condition).

The concentration of the protein in the drop in the second buffercondition (i.e., after vapor diffusion has reached equilibrium) iscontrolled by the ratio of the protein and buffer solutions in the drop.For example, two volumes of protein solution are combined with onevolume of reservoir solution to create the first buffer condition. Thus,in the first buffer condition, the concentration of reservoir solutioncomponents is 1/3 that in the reservoir solution itself. Theconcentration of protein in the first buffer condition is 2/3 of what itwas in the original protein solution. Following vapor diffusion, theconcentration of both the protein and the reservoir solution componentsin the second buffer condition will be 3 times greater than they were inthe first buffer condition. Thus, the concentration of protein in thesecond buffer condition will be approximately twice the concentration ofthe original protein solution (2/3×3).

This embodiment allows one to start with a dilute protein solution, andthen increase the concentration to a level at which the protein is notsoluble in the original protein solution. In this way, it is possible toreadily assess the ability of different buffer conditions to increasethe protein solubility above that achieved in an original proteinsolution.

For increased efficiency, it is preferable to test several differentprotein concentrations in each well by placing multiple drops, each witha different ratio of protein and buffer solutions, onto each glass slip.

A 24-well plate may be prepared using only 1-2 mL drops of a proteinsolution. This method thus affords a rapid screening of numerous bufferconditions using a minimum amount of protein.

Alternatively, vapor diffusion on a plurality of protein solutions maybe carried out using the sitting drop method or the sandwich dropmethod. Vapor diffusion apparati used in the prior art for growingcrystals simultaneously in a plurality of protein solutions may bereadily employed in the processes of the present invention. Suchapparatus are disclosed in, for example, U.S. Pat. Nos. 4,886,646,5,096,676, 5,130,105, 5,221,410 and 5,400,741, the disclosure of whichare herein incorporated by reference.

Alternatively, after combination with various buffer solutions each ofthe drops may be allowed to undergo vapor diffusion with a commonreservoir solution.

According to yet another embodiment, the process of vapor diffusion maybe repeated wherein the reservoir buffer is replaced with a buffer ofhigher osmolarity and the second buffer condition is modified to a thirdbuffer condition using vapor diffusion means. This can achieve, forexample, a stepwise concentration of protein. In yet another embodiment,this process may be repeated additional times by successively replacingthe reservoir buffer with more concentrated solutions and subjectingeach vapor-diffusion equilibrated drop to that new reservoir solution.

According to another embodiment, a volatile acid or base may be added tothe reservoir solution. Diffusion of the acid or base into the dropsmodifies the pH of the second buffer condition as compared to the firstbuffer condition.

Following vapor diffusion, the suitability of the second buffercondition for biophysical studies depends on the biophysical property tobe determined and the technique employed for that determination.Biophysical properties of a protein according to this invention includesolubility, biological activity, conformation and aggregation or foldingstate. The term "biophysical property", as used herein, refers only to aproperty that is measured when a protein is in solution. Thus, thatterm, specifically excludes techniques such as X-ray crystallography,wherein the protein is in an insoluble state.

For example, if the three dimensional structure of a protein is beinganalyzed by NMR or another spectral method, then the protein should havehigh solubility in the second buffer condition. Using the process of thepresent invention, a multitude of second buffer conditions may berapidly and efficiently screened for solubility of the protein.

The solubility of the protein in the second buffer condition canexamined by known techniques in the prior art. For example, thesolubility may be examined by visual inspection of the protein solutionfor cloudiness or precipitate. The solubility may be also be examinedsemi-quantitatively using a microscope by estimating the surface area ofthe protein solution that is covered by protein precipitate. Afterexamining different second buffer conditions, a pattern of relativesolubility and stability will typically emerge.

If the protein is soluble over a broad range of second bufferconditions, making it difficult to identify the most solubilizingconditions, then a "torture test" may be carried out by varying thetemperature of the second buffer conditions; the less stable or solublesamples will precipitate first. Alternatively, a similar test may becarried out by successively increasing the osmolarity of the reservoirsolutions following vapor diffusion and re-subjecting the drop to vapordiffusion in order to further concentrate the protein solutions.

Similarly, buffer conditions suitable for activity assays may beidentified using the processes of the present invention. For example, 20mL drops of a protein solution, each drop having a different firstbuffer condition, can be screened in batches of 24 using a 24-well plateby the sitting-drop method. From an examination of the drops after vapordiffusion, the second buffer conditions that are conducive tosolubilizing the protein can be identified by examining the drops forclarity or lack of a precipitate. From each 20 mL drop exhibitingprotein solubility, aliquots of 1 mL or less of protein solution may beprepared and tested for activity. This will allow the identification ofthe buffer condition suitable for activity assays.

Buffer conditions suitable for determining the aggregation state of aprotein in a solution may be readily identified using the processes ofthe present invention. For example, 20 mL drops of a protein solution,each drop having a different first buffer condition, can be screened inbatches of 24 using a 24-well plate by the sitting-drop method. Aftervapor diffusion, the second buffer conditions may be analyzed in adynamic light scattering instrument to determine the aggregation stateof the protein.

In spectral techniques, such as NMR, it is desirable for the proteinaggregation state to be monodisperse. In addition, the preparation ofcrystals for x-ray crystallographic analysis also requires aconcentrated protein solution, and monodisperse protein is more likelythan polydisperse protein to form high quality crystals. The presentinvention may be used to identify buffer conditions in which aconcentrated, monodisperse protein solution may be prepared prior tocarrying out a crystallization using prior art crystallographic vapordiffusion methods. Thus, the above process allows a rapid identificationof buffer conditions that are conducive for preparing monodispersesamples for spectral and crystallographic analysis.

The biophysical properties of a protein may be analyzed by applyingmultiple methods to the same drops. For example, 20 mL drops ofdifferent first buffer conditions can be screened in batches of 24 usinga 24-well plate by the sitting-drop method. After vapor diffusion, thesecond buffer condition drops may be evaluated for solubility of theprotein therein using visual examination, followed by dynamic lightscattering to determine aggregation state, and an assay to measurefunctional activity.

It will, thus, be seen that processes for identifying a second buffercondition suitable for determining a biophysical property of a proteinhave been provided. One skilled in the art will appreciate that thepresent invention can be practiced by other than the describedembodiments, which are presented for purposes of illustration and not oflimitation.

EXAMPLE 1

¹⁵ N labeled human recombinant glia maturation factor (GMF-b) with anamino-terminal His₆ tag (24 kDa) was expressed in E. coli using minimalmedia and purified using metal affinity resin at a yield of 30 mgpurified protein per liter media (Chambers, S.; Fulghum, J.; Lepre, C.,unpublished results). After refolding (Kaplan et al. 1991), the proteinwas exchanged into 50 mM potassium phosphate buffer at pH 7.4. Prior tosolvent optimization, the maximum concentration that could be achievedwas 8 mg/ml (330 mM).

After exchange into 10 mM potassium phosphate buffer, a hanging drop pHscreen was set up using a 24-well Linbro plate with siliconized glasscover slips. The reservoir volume was 1 ml of different 100 mM buffersolutions at varying pH (see Table 1 below for details), and the firstbuffer condition consisted of 2 ul of protein solution mixed with 1 ulof 100 mM reservoir buffer (final equilibrium concentration around 16mg/ml). A total of 0.384 mg of protein was used.

Following vapor diffusion 24 hours at room temperature, the amount ofprecipitate in the drops of second buffer condition was measured byplacing the trays against a black background, illuminating them from theside, and visually examining each drop under a microscope. Under theselighting conditions, precipitate appeared as a white spot against theblack background, and was scored based on the fraction of the secondbuffer condition drop covered by precipitate (scale of 0 to 4, with 0for no precipitate and 4 for precipitate completely covering the drop).The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Microdrop pH screen of GMF-b                                                      Buffer.sup.a  Drop No.     pH  Score.sup.b                                ______________________________________                                        Potassium Phosphate                                                                         1            5.0   2                                              Potassium Phosphate 2 6.0 1.5                                                 Potassium Phosphate 3 7.0 1                                                   Potassium Phosphate 4 7.4 1                                                   Sodium Phosphate 5 5.5 2.5                                                    Sodium Phosphate 6 6.5 0.5                                                    Sodium Phosphate 7 7.5 0.5                                                    Sodium Acetate 8 4.5 3                                                        Sodium Citrate 9 4.7 2.5                                                      Sodium Acetate 10 5.0 1.5                                                     Sodium Citrate 11 5.5 1.5                                                     Cacodylic Acid 12 6.5 1.5                                                     Ammonium Acetate 13 7.3 2                                                     Imidazole 14 8.0 3                                                            Bicine 15 8.5 3                                                               Bicine 16 9.0 4                                                               MES 17 5.8 3                                                                  MES 18 6.2 2.5                                                                MES 19 6.5 2                                                                  HEPES 20 7.0 1                                                                HEPES 21 8.0 1.5                                                              TRIS 22 7.5 1                                                                 TRIS 23 8.0 1.5                                                               TRIS 24 8.5 3                                                               ______________________________________                                         .sup.a all buffers are 100 mM;                                                .sup.b score is based on the surface of the drop covered by precipitate       after 24 hr at room temperature (0 = clear, i.e., fully soluble; 4 =          entire drop, i.e., insoluble).                                           

Based upon the pH screening results, it was concluded that (i) sodiumphosphate at pH 7.5 was better than the original potassium phosphatebuffer, (ii) HEPES at pH 7.0 and TRIS at pH 7.5 were good low ionicstrength alternative buffers, and (iii) MES, acetate, and any bufferwith pH ≧8.5 were poor choices.

A stabilizer screen was carried out using the most solubilizing bufferconditions identified in the pH and buffer screen: sodium phosphate atpH 7.5 and HEPES at pH 7.0. 24 drops were screened, each first buffercondition drop contained 2 ul of GMF-b (9.9 mg/ml in 10 mM potassiumphosphate) combined with 2 ul of reservoir solution. The results areshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Microdrop stabilizer screen of GMF-β                                       Stabilizers Screened Results                                                ______________________________________                                        25 mM, 50 mM, 100 mM sodium                                                                      no concentration effect                                      phosphate                                                                     10 mM beta mercaptoethanol no improvement in solubility                       5% and 10% glycerol no improvement in solubility                              HEPES at pH 7.0 less soluble than phosphate                                   2 mM CHAPS improved solubility                                                25 mM, 50 mM, 100 mM sodium no improvement in solubility                      chloride                                                                    ______________________________________                                    

Based upon these results, a new sample of GMF-24 was prepared in 50 mMsodium phosphate at pH 7.4 with 2 mM CHAPS. Using these optimized bufferconditions, the sample was successfully concentrated to 1.3 mM (afour-fold improvement from the initial solubility) and successfully usedfor NMR spectroscopy.

While I have hereinbefore presented a number of embodiments of thisinvention, it is apparent that my basic construction can be altered toprovide other embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

I claim:
 1. A process for identifying a buffer condition suitable fordetermining a biophysical property of a protein, wherein saidbiophysical property requires said protein to be in solution, comprisingthe steps of:a. providing a first buffer condition comprising a proteinand a pH-maintaining reagent, both dissolved in a solvent, b. exposingsaid first buffer condition to a reservoir solution under conditionswhich modify said first buffer condition to a second buffer conditionthrough vapor diffusion means; c. determining if said protein is solublein said second buffer condition; and d. if said protein is soluble insaid second buffer condition, evaluate the suitability of said secondbuffer condition for determining the biophysical property of theprotein.
 2. The process according to claim 1, wherein said first buffercondition further comprises a stabilizer dissolved in said solvent. 3.The process according to claim 1, wherein the concentration of theprotein in said first buffer condition is higher than the concentrationof protein in said second buffer condition.
 4. The process according toclaim 1, wherein the concentration of the protein in said first buffercondition is lower than the concentration of protein in said secondbuffer condition.
 5. The process according to claim 2, wherein saidstabilizer is selected from a salt, a reducing agent, glycerol, adetergent, a polyol, an osmolyte, a chaotrope, an organic solvent, anelectrostatic reagent, a metal ion, a ligand, inhibitor, cofactor orsubstrate, a chaperonin, a redox buffer, disulfide isomerase or aprotease inhibitor.
 6. The process according to claim 1, wherein saidbiophysical property to be determined is selected from solubility,biological activity, protein aggregation, protein folding andsuitability for NMR spectroscopy.
 7. The process according to claim 1,wherein the pH of said first buffer condition differs from that of saidsecond buffer condition.
 8. The process according to claim 1, wherein instep b), multiple first buffer conditions are exposed simultaneously tothe same reservoir solution.
 9. The process according to claim 1,wherein in step b), multiple first buffer conditions are exposedsimultaneously to multiple reservoir solutions.